Cytokine-induced changes in chromatin structure and in vivo footprints in the inducible NOS promoter.

Transcription of the human inducible nitric oxide synthase (iNOS) gene is regulated by inflammatory cytokines in a tissue-specific manner. To determine whether differences in cytokine-induced mRNA levels between pulmonary epithelial cells (A549) and hepatic biliary epithelial cells (AKN-1) result from different protein or DNA regulatory mechanisms, we identified cytokine-induced changes in DNase I-hypersensitive (HS) sites in 13 kb of the iNOS 5'-flanking region. Data showed both constitutive and inducible HS sites in an overlapping yet cell type-specific pattern. Using in vivo footprinting and ligation-mediated PCR to detect potential DNA or protein interactions, we examined one promoter region near -5 kb containing both constitutive and cytokine-induced HS sites. In both cell types, three in vivo footprints were present in both control and cytokine-treated cells, and each mapped within a constitutive HS site. The remaining footprint appeared only in response to cytokine treatment and mapped to an inducible HS site. These studies, performed on chromatin in situ, identify a portion of the molecular mechanisms regulating transcription of the human iNOS gene in both lung- and liver-derived epithelial cells.

Pulmonary function response to EDTA, an additive in nebulized bronchodilators.

BACKGROUND: Some nebulized bronchodilator solutions contain additives, such as EDTA, benzalkonium chloride (BAC), or both. OBJECTIVE: Although BAC-induced bronchoconstriction has been well documented in patients with asthma, there is no information on the effects of EDTA on FEV(1) when inhaled in the amounts that would be administered during emergency department treatment of asthma. METHODS: Eighteen subjects with stable asthma and airway responsiveness to methacholine were randomly assigned to inhale up to four 600-microg nebulized doses of EDTA, BAC (positive control), and normal saline (placebo) in a double-blind crossover manner on separate days. FEV(1) was measured 15 minutes after each dose. Treatments were repeated every 20 minutes until FEV(1) decreased by 20% or greater or a maximum of 4 doses were administered. RESULTS: Mean +/- SD maximum percent decrease in FEV(1) was 1.8% +/- 5.8% after EDTA, 16.6% +/- 13.9% after BAC, and 3.6% +/- 8.2% after placebo (P <.001); there was no significant difference between EDTA and placebo. CONCLUSION: The amount of EDTA contained in maximum recommended doses of nebulized bronchodilators does not induce bronchospasm. In contrast, BAC induces clinically important bronchospasm, which could decrease the efficacy of a bronchodilator during an emergency.

Resolution of hypoxemia in a liver transplant recipient after ligation of a portosystemic shunt.

This report describes the unique development of pulmonary vascular dilatation and hypoxemia associated with a portosystemic shunt in a pediatric liver transplant recipient. Ligation of the shunt resulted in resolution of hypoxemia. The outcome suggests that hepatic venous return to the pulmonary circulation is important in maintaining normal pulmonary vascular caliber.

Cytokine-inducible enhancer with promoter activity in both the rat and human manganese-superoxide dismutase genes.

Diverse pro-inflammatory mediators regulate transcription of the gene (MnSOD) encoding the mitochondrial anti-oxidant protein manganese-superoxide dismutase. Understanding the regulation of this gene is crucial to comprehending its role in cytoprotection. In transfected lung epithelial cells, a human-growth-hormone reporter gene system was utilized to identify a potential enhancer in the MnSOD genomic fragment previously shown to contain multiple DNase-I-hypersensitive sites. Northern analysis demonstrated a 10-20-fold increase in response to pro-inflammatory mediators. Inclusion of the MnSOD genomic fragment in reporter constructs was necessary to mimic these stimulus-dependent endogenous levels. The inducible enhancer element was localized to a 260 bp fragment in intron 2, coinciding with a previously defined DNase-I-hypersensitive site. This element functions in an orientation- and position-independent manner as well as with the heterologous thymidine kinase promoter. In addition, we have demonstrated that a homologous sequence within the human MnSOD gene exhibits identical enhancer activity. A novel characteristic of the rat and human enhancer elements involves the ability to promote cytokine-inducible transcription in the absence of a classical promoter.

In vivo architecture of the manganese superoxide dismutase promoter.

Mitochondrial manganese superoxide dismutase (Mn-SOD) is the primary cellular defense against damaging superoxide radicals generated by aerobic metabolism and as a consequence of inflammatory disease. Elevated expression of Mn-SOD therefore provides a potent cytoprotective advantage during acute inflammation. Mn-SOD contains a GC-rich and TATA/CAAT-less promoter characteristic of a housekeeping gene. In contrast, however, Mn-SOD expression is dramatically regulated in a variety of cells by numerous proinflammatory mediators, including lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1. To understand the underlying regulatory mechanisms controlling Mn-SOD expression, we utilized DNase I-hypersensitive (HS) site analysis, which revealed seven hypersensitive sites throughout the gene. Following high resolution DNase I HS site analysis, the promoter was found to contain five HS subsites, including a subsite that only appears following stimulus treatment. Dimethyl sulfate in vivo footprinting identified 10 putative constitutive protein-DNA binding sites in the proximal Mn-SOD promoter as well as two stimulus-specific enhanced guanine residues possibly due to alterations in chromatin structure. In vitro footprinting data implied that five of the binding sites may be occupied by a combination of Sp1 and gut-enriched Kr uppel-like factor. These studies have revealed the complex promoter architecture of a highly regulated cytoprotective gene.

Regulation of the manganese superoxide dismutase and inducible nitric oxide synthase gene in rat neuronal and glial cells.

Bidirectional communication occurs between neuroendocrine and immune systems through the action of various cytokines. Responses to various inflammatory mediators include increases in intracellular reactive oxygen species (ROS), notably, superoxide anion (O2-) and nitric oxide (NO.). Neurotoxicity mediated by NO. may result from the reaction of NO. with O2, leading to formation of peroxynitrite (ONOO-). ROS are highly toxic, potentially contributing to extensive neuronal damage. We, therefore, evaluated the effects of a variety of inflammatory mediators on the regulation of mRNA levels for manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in primary cultures of rat neuronal and glial cells. To determine age-dependent variation of mRNA expression, we used glial cells derived from newborn, 3-, 21-, and 95-day-old rat brains. Interleukin-1 beta, interferon-gamma (IFN-gamma), bacterial lipopolysaccharide (LPS), and tumor necrosis factor-alpha showed significant induction of MnSOD in both glial and neuronal cells. However, only LPS and IFN-gamma increased iNOS mRNA. These data demonstrate that these two genes are similarly regulated in two cells of the nervous system, further suggesting that the oxidative state of a cell may dictate a neurotoxic or neuroprotective outcome.

Isolation and maintenance of human pulmonary artery endothelial cells in culture isolated from transplant donors.

Even though endothelial cells from different locations have similarities, there are potential morphological and functional differences between cells from different vascular regions, as well as between species. Our laboratory is interested in studying the molecular regulation of vasoactive substances in pulmonary vasculature. Therefore, we have developed reproducible methodology to isolate and maintain cultures of human pulmonary artery endothelial cells. The major innovation has been the employment of sections of pulmonary artery from heart transplant donors, from which endothelial cells are isolated. Cell monolayers were identified as endothelial cells by phase-contrast microscopy. Representative dishes of cells were further characterized by indirect immunofluorescent staining for factor VIII antigen, uptake of acetylated low-density lipoprotein, and electron microscopy. These cells were also evaluated for the expression of endothelin-1 (ET-1), a vasoactive 21-amino acid peptide derived from endothelial cells. The cells expressed ET-1 peptide and mRNA as determined by radioimmunoassay and Northern analysis, respectively. We also demonstrated that these cells are useful in transient transfection experiments for potential evaluation of promoter elements. The availability and relevance of these cells provide an important investigative tool for studies on human pulmonary vascular disease.

Regulation of inducible nitric oxide synthase mRNA levels by LPS, INF-gamma, TGF-beta, and IL-10 in murine macrophage cell lines and rat peritoneal macrophages.

The molecular mechanisms of LPS, INF-gamma, TGF-beta, and IL-10 regulation of inducible nitric oxide synthase (iNOS) mRNA expression were evaluated. In murine macrophage cell lines, LPS-induced increases in iNOS mRNA were blocked by either cycloheximide or actinomycin D. Neither TGF-beta nor IL-10 alone had any effect on basal expression, and each only slightly reduced LPS induction of iNOS mRNA. However, IL-10 augmented INF-gamma induction of iNOS mRNA to very high levels, while TGF-beta inhibited INF-gamma induction. Human monocytes expressed no detectable iNOS mRNA with any stimuli, though Southern analysis on human genomic DNA revealed a specific human iNOS gene. In human macrophages, the iNOS gene may have become inoperative during evolution.

Regulation of manganese superoxide dismutase: IL-1 and TNF induction in pulmonary artery and microvascular endothelial cells.

IL-1 and TNF are important mediators in the inflammatory response, and have been associated with endothelial cell damage in the lung. TNF and IL-1 cell-mediated injury has been proposed to occur through an increase in intracellular oxygen free radical production. However, these cytokines have also been shown to protect the lung from hyperoxia-mediated oxidant injury. In this paper we evaluated the response of the antioxidant enzymes, MnSOD and Cu/ZnSOD to IL-1, TNF, and LPS in both rat pulmonary artery and microvascular endothelial cells. These mediators produced an increase in MnSOD but not Cu/ZnSOD expression in both rat pulmonary endothelial cells. An additive effect was observed with co-treatment by the cytokines with LPS. The MnSOD mRNA induction is dependent upon a transcriptional event, but did not require de novo protein synthesis.

Clinical application of transepithelial potential difference measurements in cystic fibrosis.

We studied transepithelial potential difference (PD) in normal persons, patients with chronic disease, and patients with cystic fibrosis (CF), using the technique described by Knowles and co-workers. A maximal PD value (PDmax) and an average PD value (PDmean) were determined for each study of the nasal respiratory epithelium. The voltage response to superfusion of 10(-4) mol/L amiloride onto nasal mucosa was noted. The PD of the palm, wrist, and between two fingertips was also measured. Nasal PDmax and PDmean of the CF group were more negative than the control (P less than 0.01) and chronic disease groups (P less than 0.01). After application of amiloride, the voltage change in nasal PD was greater in the CF group than in the non-CF control groups (P less than 0.01). There were no clinically significant differences in the PD of the palm, wrist, or fingertips of the three groups. These data confirm the observation that patients with CF have hyperpolarized nasal epithelia that demonstrate greater change in response to amiloride than that in non-CF controls. These results indicate a possible role for the use of in vivo nasal PD measurements as a diagnostic test for cystic fibrosis.

Effect of massage on serum level of beta-endorphin and beta-lipotropin in healthy adults.

We conducted this study to evaluate the effect of massage on the levels of endogenous opiates in peripheral venous blood. The results were based on findings from 21 healthy, adult volunteers. After separation by sex, the volunteers were assigned randomly to either the Control Group (n = 11) that rested but received no massage or the Experimental Group (n = 10) that received a 30-minute complete back massage. We found no significant pretreatment or posttreatment difference in blood beta-endorphin or beta-lipotropin levels between the groups. The results indicate that massage did not change significantly the measured serum levels of beta-endorphin or beta-lipotropin in our healthy subjects without pain. A follow-up study using patients experiencing acute or chronic back pain is recommended. Massage is used routinely in the treatment of such patients, and endogenous opiates are recognized as a possible mechanism for pain relief.

Total lung lavage for pulmonary alveolar proteinosis in an infant without the use of cardiopulmonary bypass.

Pulmonary alveolar proteinosis is a rare disease that usually affects the adult patient, but is now being recognized as a possible cause of neonatal respiratory distress. In the adult patient, whole lung lavage, as described by Ramirez-R in 1965, is considered the most effective therapy for management of this condition. The lavage can be accomplished safely and with relative ease by using a Carlens or Robertshaw tube to isolate and lavage one lung while ventilating the other. The unavailability of a small double-lumen tube makes this procedure impossible in the pediatric age group. Therefore, whole lung lavage has been possible in only a few children in the past with the help of cardiopulmonary bypass to allow simultaneous oxygenation during the pulmonary lavage. Due to the hazards and technical difficulties of cardiopulmonary bypass, total pulmonary lavage can not be considered a practical option in the very small infant. A 15-week-old infant is reported, weighing 2 kg with a diagnosis of pulmonary alveolar proteinosis, who underwent total pulmonary lavage safely on three different occasions without employing cardiopulmonary bypass. A double-lumen Swan-Ganz catheter, introduced transbronchoscopically through the side-arm of a rigid, 3.5-mm Storz bronchoscope was used to isolate and lavage one lung while ventilation to the other lung was maintained through the bronchoscope. A Nellcor oximeter, utilized for transcutaneous monitoring, revealed satisfactory oxygen saturation during the entire pulmonary lavage. The transbronchoscopic lavage was monitored under direct vision with a video monitor, ensuring correct position of the bronchoscope and the catheter at all times.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunologic release of histamine from dog lung: comparison of in vivo and in vitro responses in the same animal.

The purpose of this study was to compare, for the first time, antigen-induced histamine release from the lung in the same natively allergic dogs both in vitro and in vivo. In six dogs, maximal antigen-induced histamine release from the lung correlated closely in vitro and in vivo (r = 0.94), although it varied widely between dogs (0% to 75.5% of total tissue histamine content); similarly, the antigen concentration to produce 50% of maximal histamine release varied sixfold between dogs (40 micrograms/ml to 250 micrograms/ml). In each of five other dogs, terbutaline sulfate administered intravenously caused a dose-dependent inhibition of antigen-induced histamine release from lung fragments in vitro: the maximal inhibition produced by 1 mg/kg was 60 +/- 4.5% (mean +/- SEM). In these same dogs, 10(-5)M terbutaline incubated with lung fragments in vitro caused inhibition of antigen-induced histamine release comparable to 1 mg/kg terbutaline in vivo. Increasing the dose of terbutaline in vitro produced maximal inhibition at 10(-4)M with no greater effect of the drug at 10(-3)M (71.4 +/- 3.8% inhibition). In both experimental situations propranolol caused a dose-dependent inhibition of beta-adrenergic modulation of Ascaris-induced release of histamine. This result supports the conclusion that terbutaline produced its effects by actions mediated by beta-adrenergic receptors on pulmonary mast cells. This experimental approach provides a suitable preparation in which to estimate the effective dose of agonists that modulate antigen-induced mast cell function in vivo.

Pulmonary function in insulin-dependent diabetes mellitus with limited joint mobility.

Patients with insulin dependent diabetes mellitus (IDDM) and limited joint mobility (LJM) were studied to determine if altered respiratory mechanics were another manifestation of a generalized disturbance in collagen metabolism. Lung volumes and maximal expiratory flow volume curves were measured in 23 patients with IDDM. Patients were divided into 2 groups: (1) 11 without LJM, and (2) 12 with severe LJM. The groups were matched for age, sex, and glycemic control but not for duration of IDDM. In patients with severe LJM, forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) were significantly decreased (p less than 0.05). Total lung capacity (TLC), thoracic gas volume (TGV) at functional residual capacity (FRC) and residual volume (RV) were also significantly lower (p less than 0.05) in the severe LJM group. There was no evidence of air-flow obstruction in either group. Our results demonstrate an association between severe LJM and a significant decrease in lung volumes. This could be due to decreased lung compliance or restriction of chest wall expansion.

Effect of beta-adrenergic stimulation on experimental canine anaphylaxis in vivo.

We studied beta-adrenergic modulation of antigen-induced release of histamine and changes in lung function in natively allergic, anesthetized dogs. After thoracotomy and bilateral cervical vagotomy, samples of peripheral lung frozen in situ were obtained for measurement of adenosine cyclic 3',5'-monophosphate (cAMP) and histamine. In 11 dogs with beta-adrenergic blockade (propranolol), subsequent Ascaris suum antigen (intravenous) decreased lung tissue histamine 21.5% +/- 5.3% (mean +/- SE), with marked physiologic changes of anaphylaxis. In six other dogs, with no pharmacologic treatment before antigen, A. suum decreased lung tissue histamine only 11.5% +/- 1.9%. In six other dogs, terbutaline (1 mg/kg, i.v.) increased concentration of cAMP in peripheral lung, indicating stimulation of beta-adrenergic receptors, and inhibited response to A. suum. When a beta-adrenergic agonist was given 15 sec prior to A. suum, cAMP doubled and inhibition was incomplete. When a beta-agonist was given 150 sec prior to A. suum, cAMP increased fivefold and inhibition was complete.

In vivo demonstration of nonadrenergic inhibitory innervation of the guinea pig trachea.

To determine if electrical stimulation of autonomic nerves could excite nonadrenergic inhibitory motor pathways in the guinea pig respiratory system in vivo, we studied the effects of electrical stimulation of the cervical vagi and sympathetic nerve trunks on pressure changes (P(p)) within an isolated, fluid-filled cervical tracheal segment which reflected changes in trachealis muscle tone. We preserved the innervation and circulation of the segment as evidenced by a rise in P(p) with vagus nerve stimulation and a fall in P(p) with intravenous isoproterenol. In five atropine-treated animals, stimulation of the cut vagi or sympathetic nerve trunks resulted in a mean fall in P(p) of 7.9 and 8.2 cm H(2)O, respectively. Treatment with propranolol attenuated the response to sympathetic stimulation but not vagal stimulation. To determine if these relaxation responses were mediated by an adrenergic or nonadrenergic mechanism, we studied an additional five animals that had been treated with 6-hydroxydopamine to destroy adrenergic nerve endings. In 6-hydroxydopamine, atropine, and propranolol-treated animals, sympathetic nerve stimulation decreased P(p) only 0.65 cm H(2)O, confirming the elimination of adrenergic nerve influences, whereas vagus nerve stimulation decreased P(p) 17.7 cm H(2)O. After sectioning the recurrent laryngeal nerves, the mean decrease in P(p) during vagus nerve stimulation was only 3.2 cm H(2)O. These findings demonstrate the presence of nonadrenergic inhibitory nerves in the guinea pig trachea in vivo. They further show that nonadrenergic inhibitory nerve effects are elicited during electrical stimulation of the vagus nerves and that interruption of the recurrent laryngeal nerves diminishes the magnitude of these effects.

Experimental canine anaphylaxis: cyclic nucleotides, histamine, and lung function.

We studied the effects of Ascaris suum antigen (iv) in 32 natively allergic, anesthetized dogs. After thoracotomy, bilateral cervical vagotomy, and propranolol, samples of peripheral lung frozen in situ were obtained for measurement of cyclic nucleotides and histamine. Following Ascaris, lung histamine decreased 20.4 +/- 3.7% (mean +/- SE), cAMP increased 391 +/- 122%, and cGMP increased 110 +/- 20% with increased plasma histamine and physiological changes of anaphylaxis. No significant changes occurred in 10 control dogs. Release of histamine, reflecting immunological degranulation of mast cells, correlated closely with the physiological effects of anaphylaxis. beta-Adrenergic stimulation with isoproterenol prevented these physiological effects of anaphylaxis in three dogs studied. Furthermore, the level of cAMP induced in lung tissue by beta-adrenergic stimulation in these dogs correlated with the degree of inhibition of immunologically induced histamine release. These results illustrate the suitability of this experimental preparation to study the biochemical and physiological mechanisms of anaphylaxis in vivo.